Transplantation of neuronal and glial restricted precursors into contused spinal cord improves bladder and motor functions, decreases thermal hypersensitivity, and modifies intraspinal circuitry

Abstract

Transplanting neuronal and glial restricted precursors (NRP/GRP) into a midthoracic injury 9 d after contusion improved bladder and motor function, diminished thermal hypersensitivity, and modified lumbosacral circuitry compared with operated controls (OP-controls). Histological analysis showed that NRP/GRP survived, filled the lesion site, differentiated into neurons and glia, and migrated selectively. Volume of spinal cord spared was increased in NRP/GRP recipients, suggesting local protection. Bladder areflexia developed in both operated groups, but NRP/GRP recipients exhibited an accelerated recovery, with decreased micturition pressure and fewer episodes of detrusor hyperreflexia. Because noradrenergic receptors proliferate after spinal injury and descending noradrenergic pathways contribute to regulation of bladder control, we examined the effects of administering an alpha-1A-adrenergic antagonist, Tamsulosin, on urodynamics. This improved all cystometric parameters in both operated groups, and micturition pressure in NRP/GRP rats recovered to normal levels. Both operated groups initially showed increased sensitivity to a thermal stimulus applied to the tail; the NRP/GRP rats showed significant improvement over time. NRP/GRP grafts also produced greater recovery of hindlimb function in several tests, although both groups showed persistent and similar deficits in locomotion on a grid. Because bladder, hindlimb, and tail sensory and motor functions are organized through lumbosacral cord, we examined descending and primary afferent projections at L6-S1. The density of serotonergic, noradrenergic, and corticotrophin releasing factor-positive fibers increased in the NRP/GRP group compared with OP-controls, suggesting some sparing and/or sprouting of these modulatory pathways. Immunocytochemical staining density of dorsal root axons in the dorsal horn increased in the OP-controls but appeared normal in the NRP/GRP group. Synaptophysin immunoreactivity in the lumbosacral dorsal horn was similar among groups, consistent with restoration of synaptic density in both groups of operated animals but by different pathways. We suggest that local protection provided by NRP/GRP resulted in increased sparing/sprouting of descending pathways, which prevented sprouting by dorsal root axons, and that this modification in lumbosacral circuitry contributes to the recovery of function.

Figure 1.

Micturition behavior in metabolic cages. The voided volume per micturition significantly increased in NRP/GRP recipients (n = 8) 2 weeks after transplantation (^*p < 0.05) compared with OP-controls (n = 10). OP-controls reached the same level as NRP/GRP rats at week 4. Thus, NRP/GRP recipients showed an acceleration of recovery from areflexic bladder.

Figure 2.

Representative cystometry charts of unoperated control, OP-control, and NRP/GRP rats. A, Rhythmic contractions (↓) that do not result in voiding are not seen in unoperated control rats. Tamsulosin administration does not modify these patterns in normal rats. B, When vehicle is infused into the bladder, rhythmic intravesical pressure waves (↓) appear before voiding (^*). In OP-control rats, frequent rhythmic contractions occur, and these are only rarely associated with voiding (^*). This indicates hyperactivity of the bladder (DHR). Micturition pressure (^*) was higher compared with unoperated controls, indicating DSD. Tamsulosin administration decreases micturition pressure and the number of contractions that are not associated with voiding. C, NRP/GRP rats show lower micturition pressure (^*) and few contractions that are not associated with voiding (DHR) (↓) after saline infusion compared with OP-controls. Tamsulosin administration slightly decreases micturition pressure and the number of reduced contractions that are not associated with voiding.

Figure 3.

Amelioration of hypersensitivity to thermal stimuli and recovery of motor function (^*p < 0.05; ^**p < 0.01). A, Latency of withdrawal to thermal stimulus. The latency for tail withdrawal was decreased in both groups compared with baseline, but the NRP/GRP group showed significantly attenuated responses compared with OP-controls. B, Hindlimb function. Hindlimb motor function was assessed using the open-field BBB scoring system. NRP/GRP transplanted rats showed improvement in BBB scores beginning at week (wk) 4 and maintained through week 8 compared with OP-controls. C, Grid test. Both operated groups showed sharply increased errors on the grid at 1 week after transplantation, with no functional improvement and no differences between the OP-control and NRP/GRP recipients. TP, Transplantation.

Figure 4.

Nissl-myelin staining and immunocytochemistry at the lesion site. Dotted lines indicate lesion border. A, Longitudinal section of spinal cord from OP-control rat. The injury site is filled with the Vitrogen matrix, into which some cells have migrated (^*, connective tissue; H, host). B, Longitudinal section of spinal cord in Nissl-myelin staining shows survival of NRP/GRP transplanted into the injured spinal cord at 8 weeks after transplantation. The dotted line indicates graft-host interface. The cavity was primarily filled with transplanted cells, and few cysts were apparent (H, host; G, graft). C, Few RT-97⁺ axons were found within the Vitrogen matrix in OP-controls. D, In contrast, RT-97 axons were densely distributed within the graft in NRP/GRP. E, In OP-controls, many GFAP⁺ cells surrounded the lesion site, contributing to a glial scar. F, In NRP/GRP recipients, many GFAP⁺ cells fill the transplant. G, RIP⁺ cells were diminished adjacent to the lesion site in OP-controls, indicating demyelination. H, NRP/GRP recipients contained many RIP⁺ cells within the graft.

Figure 5.

Confocal microscopy of immunocytochemical staining for phenotypes of transplanted cells, and axonal staining and synaptophysin immunoreactivity within the grafts. A, Section of spinal cord adjacent to Figure 4 histochemically stained for AP to identify grafted cells. Injection sites were rostral to the lesion (1), at the lesion epicenter (2), and caudal to the lesion (3). The injection site is filled with AP⁺ cells. AP⁺ cells also migrated into the parenchyma and rostrally and caudally to the injection sites. Inset shows AP⁺ cells at the rostral extents of migration into host tissue. B, C, Neuronal differentiation was assessed by colocalization of NeuN (red; B) and MAP2 (red; C) with the AP transgene (green) (NeuN⁺/AP⁺ and MAP2⁺/AP⁺ cells, arrows). Scale bars, 20 μm. D, Astrocytic differentiation was assessed by colocalization of GFAP (red) with AP (green) (GFAP⁺/AP⁺ cells, arrow). Scale bar, 20 μm. E, Oligodendrocytic phenotype was shown by colocalization of RIP (red) with AP (green) (RIP⁺/AP⁺ cells, arrow). Scale bar, 20 μm. F, Synaptophysin immunoreactivity. Abundant synaptophysin immunoreactivity was seen within the grafts, suggesting formation of presynaptic terminals and possible formation of synaptic connection within the graft and with the host.

Figure 6.

Primary afferent targets. Scale bars, 100 μm. A, Distribution of CGRP immunoreactivity in the superficial layers of the DH was similar in unoperated controls and NRP/GRP. Immunoreactivity of CGRP appeared denser in OP-controls (open arrows). B, GAP-43 immunoreactivity in the DH was similar in unoperated controls and NRP/GRP rats and denser in OP-controls (open arrow). GAP-43 was greatly diminished in the CST (filled arrow) of both operated groups compared with the unoperated control rats. C, Synaptophysin immunoreactivity is similar among groups.

Figure 7.

Quantification of immunoreactivity (^*p < 0.05, ^** p < 0.01 in OP-controls vs NRP/GRP; †p < 0.05, ††p < 0.01 in OP-controls vs unoperated-Controls; ‡‡p < 0.01 in NRP/GRP vs unoperated controls). A, CGRP immunoreactivity in the DH was significantly denser in OP-controls than in NRP/GRP and unoperated controls, but there were no differences in DCM among groups. B, VR-1 immunoreactivity was significantly denser in OP-controls than in NRP/GRP and unoperated controls, but there were no differences in DCM among groups. C, GAP-43 immunoreactivity was significantly increased in the DH in OP-controls compared with NRP/GRP and unoperated controls. GAP-43 immunoreactivity in the CST was significantly less in both operated groups compared with unoperated controls. D, There were no differences in synaptophysin immunoreactivity among the groups. E, 5-HT axons in the DL and DH were more densely distributed in NRP/GRP compared with OP-control rats, although less than in unoperated control rats, and serotonergic fibers were considerably more densely distributed in the lateral funiculus (LF) adjacent to the DL in the NRP/GRP group than in either unoperated control or OP-control groups. F, CRF-positive fibers in the SPN of NRP/GRP rats were intermediate in density between unoperated control and OP-control groups. Many CRF-positive fibers were identified in the DH of NRP/GRP and some CRF fibers were observed in OP-controls compared with little or no CRF immunoreactivity in unoperated control rats.

Figure 8.

DβH-positive fibers in lumbosacral cord. Scale bars, 100 μm. A, DβH immunoreactivity was densest in the DL in unoperated controls and least dense in OP-controls. Note staining of motor neurons in all groups. B, DβH immunoreactivity was densest in the SPN in unoperated controls and least dense in OP-controls. C, In the DH, DβH immunoreactivity was diminished in NRP/GRP, but only a few DβH fibers were seen in OP-controls.